P478: THE GERMLINE VARIANT GFI1-36N PROMOTES GENETIC INSTABILITY IN LEUKEMIC CELLS AND OPENS NEW THERAPEUTIC APPROACHES FOR ABOUT 15% OF ALL AML PATIENTS
Authors: J. Vorwerk, D. Frank, K. Sun, F. Neumannn, M. Heuser, D. Kunadt, W. E. Berdel, J.-H. Mikesch, C. Schliemann, G. Lenz, A. K. Jayavelu, C. Khandanpour
Journal: HemaSphere (2022)

Background: The zinc finger protein Growth Factor Independence 1 (GFI1) acts as a transcriptional repressor regulating differentiation of myeloid and lymphoid cells. In a single-nucleotide polymorphism (SNP) of GFI1, GFI1-36N, the amino acid serine (S) is replaced by asparagine (N) at position 36. GFI1-36N has a prevalence of 7% in healthy Caucasians and 15% in acute myeloid leukemia (AML) patients, hence predisposing to AML. We have shown previously that this is particularly since the GFI1-36N protein differs from the GFI1-36S variant regarding its ability to induce epigenetic changes leading to a derepression of oncogenes. Now we have evidence that GFI1 is implicated in DNA repair as AML blast cells of patients carrying the GFI1-36N SNP feature more frequently complex-aberrant karyotypes and more mutations leading to an inferior prognosis. Aims: Therefore, in this work, we explored whether presence of GFI1-36N alters DNA repair activity and whether this can be targeted therapeutically. Methods: We analyzed the association between GFI1-36N and chromosomal aberrations in human AML samples. As a murine model of human AML, we knocked in the human GFI1-36S or GFI1-36N variant into the murine Gfi1 gene locus and retrovirally expressed MLL-AF9 to induce AML. We then performed γ-H2AX immunofluorescence and flow cytometry to quantify the number of DNA double-strand breaks (DSBs), confocal immunofluorescence microscopy to detect DNA repair foci of key enzymes of non-homologous end joining (NHEJ) and homologous recombination (HR) as well as proteomics and immunoblotting to determine the level of DNA damage response enzymes and cell cycle kinases. Cell viability assay and colony-forming unit assay were used to examine the response of GFI1-36S and GFI1-36N leukemic cells to palbociclib treatment. Results: A complex aberrant karyotype was found twice as often in patients carrying the GFI1-36N SNP compared to GFI1-36S controls (38% vs. 20%, p** = 0.0080). Murine GFI1-36N-MLL-AF9 cells showed 43 times more genetic and cytogenetic aberrations than GFI1-36S-MLL-AF9 controls (p*** = 0.0003). Upon exposure to irradiation, murine GFI1-36N leukemic cells featured more DSBs and delayed repair. Regarding the two main DSB repair pathways, we did not see a significant difference in the ability of GFI1-36N or GFI1-36S leukemic cells to perform NHEJ, but a reduced HR capacity in presence of GFI1-36N. On a molecular level, GFI1-36N was associated with less foci of the HR key enzyme RAD51, reduced protein levels of the DNA damage response enzymes CHK1 and CDK2, higher levels of the cell cycle propagating CDKs, and reduced levels of the CDK inhibiting CDKNs. As especially CDK4 and CDK6 protein levels were significantly increased in GFI1-36N leukemic cells, we subsequently treated malignant cells with the established CDK4/6 inhibitor palbociclib. We observed that GFI1-36N leukemic cells were more susceptible to this treatment, indicated by 49% less CFUs compared to GFI1-36S leukemic controls (p** = 0.0063). Image:Summary/Conclusion: The findings suggest that the GFI1-36N SNP increases cell cycling and reduces DNA repair activity. This could promote emergence of chromosomal aberrations and hence contribute to genomic instability of leukemic cells (Figure 1). On a therapeutic level, GFI1-36N could possibly be a marker for a specific subset of AML patients that is sensitive to CDK4/6 inhibitors.